On the Effect of Anthranilic Acid. (2)

View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Kyoto University Research Information Repository Studies on the Biosynthesis of Pyocyanine. (VIII) : On the Title Effect of Anthranilic Acid. (2) Author(s) Kurachi, Mamoru Bulletin of the Institute for Chemical Research, Kyoto Citation University (1959), 37(2): 101-111 Issue Date 1959-07-10 URL http://hdl.handle.net/2433/75698 Right Type Departmental Bulletin Paper Textversion publisher Kyoto University Studies on the Biosynthesis of Pyocyanine. (VIII) On the Effect of Anthranilic Acid. (2) MamoruKURACHI* (KatagiriLaboratory) ReceivedFebruary 4, 1959 As an evidencesupporting the possibilityof anthranilic acid to bean intermediatein pyocyaninesynthesis, it was foundthat anthranilicacid was accumulatedin the medium of bacteriumPseudomonas aeruginosa, cultured in the presenceof inhibitoryagent, and that pyocyanine could be synthesizedfrom anthranilicacid in resting system,by ad- ministeringglutamic acid or glycine.It has beenresulted that theinhibitory agent such as aniline,o-phenylenediamine and p-aminobenzoicacid shouldbe a metaboliteanalogue of anthranilicacid. On the other hand,anthranilic acid has been demonstratedto be formedthrough bothanabolic and catabolicpathways, and postulatedto be, in part, deriveddirectly from indoleby oxidativecleavage of its nucleusas a reversereaction of the conversion of anthranilicacid to indole. INTRODUCTION In the precedingwork'' designed according to the conceptthat the aromatic compoundrevealing an inhibitoryaction on pyocyanineformation may be a metaboliteanalogue of the intermediatein pyocyaninesynthesis, anthranilic acid has been pointedout to be a possibleintermediate in this system. Howeverthe experiment was unsuccessfulin the synthesis of pyocyanine under an enzymatic condition, and yet it has been shown that the reaction product from anthranilic acid by resting cells was also regarded as the intermediatein pyocyaninesynthesis from the fact that this product was possiblyidentical with the one accumulatedin the medium of the bacteria which has been considered to be an intermediate in pyocyanine synthesis. Provided the concept mentionedabove is reasonable, the ac- cumulationof anthranilic acid should be brought about in the presence of the inhibitoryagent. Based on the fact that the present strain can grow on the synthetic medium prepared with urea as a sole source of nitrogen, anthranilicacid must be regarded to be synthesized by the bacteria. However the work dealing with the formation of anthranilic acid by Pseudomonasaeruginosa had not been presented. Recently,Takeda and Nakanishi2'have reported that anthranilic acid was isolated from the culture medium of the bacteria mentionedabove. In the present work, the experiment has succeeded in obtaininganthranilic acid from the synthetic medium containingthe inhibitoryagent whichhad preced- (1_01) Mamoru KURACHI ingly been put forward, and demonstrated that the inhibitors such as aniline or acetanilide, o-phenylenediamine and p-aminobenzoic acid are metabolite analogue of anthranilic acid. On the other hand, it has been known that when phosphate was kept at an extremely low level, pyocyanine could enzymatically be synthesized from anthranilic acid in the presence of glutamic acid or glycine. This condition was found to be also applicable to the other case of the enzymatic synthesis of pyocyanine. EXPERIMENTAL AND DISCUSSION Formation of Anthranilic Acid In order to find out the condition for the formation of anthranilic acid, two kinds of the medium containing the following materials were prepared : (1) 2% glycerol, 1% peptone, 0.2% urea, 0.05% MgSO47H2O and 0.025V K2HPO4. (2) 2%o glycerol, 0.2% urea, 0.050/ MgSO47H2O, 0.025% K2HPO4 and 0.0005% Fe2(SO4)3. With the above media, various strains of Pseudomonas aeruginosa were cultured at 3T for 48 hours. The cultured solution was acidified with dilute sulfuric acid and extracted with ethyl ether to test their fluorescence under ultraviolet light. As shown in Table 1, with the peptone medium fluorescence Table 1. Formation of fluorescent product. Fluorescence MediumStrains B1 B2 Bk C1 Bt Bd No. 1+ + Trace + + + No. 241ff ifF 14- 11+ +If iff No. 1 : 2% glycerol, 0.2% urea, 0.05% MgSO47H2O, 0.025% K2HPO4, and 0.0005% Fe2(SO4)3, pH 7.4. No. 2: 1% peptone was added to No. 1. Incubation, at 37° for 48 hours. Fluorescence was observed with ether extract of the cultured solution. was considerably revealed in major cases of the strain, while with the synthetic medium it was hardly indicated. In addition to this experiment, effects of various inhibitory agents were tested on the detection of the fluorescent product. As was previously mentioned," a discrepancy is shown in a considerably wide range between the limiting concentrations for the growth and for the pigmentation, so that the inhibitory agent was given at a maximal concentration at which the bacterial growth was satisfactory while no pyocyanine was formed. The inhibitors dissolved in alcohol were added to the synthetic medium shown above after its pasteurization and incubated at 3T for 3 days. The results are shown in Table 2. Among the cultured solutions of various inhibiting agents, it was observed that fluorescence was remarkably exhibited in the case in which aniline (or acetanilide), o-phenylenediamine or p-aminobenzoic acid was added to the medium as an inhibitor, whereas in the case of polyphenols results were negative with the exception of resorcinol, of which fluorescence was found to originate (102) Studies on the Biosynthesis of Pyocyanine. (VIII) Table 2. Formation of flurorescent product. Inhibitory agents addedFluorescenceSpot N o. 1No. 2 corresponding to AA Aniline ~fif jIffI Acetanilidedl$I}+ o-Phenylenediaminedldl+ P-Aminobenzoic aciddl$fF+ Phenol-FE+— Resorcinoldi-dl— Pyrocatechol}F+— Hydroquinone}F+— Guajacol-I1-+— Control-I 1+— Basal medium was the same as No. 1 in Table 1. No. 1, the result with butanol extract ; No. 2, ether extract. AA, anthranilic acid. from itself during the preservation of its solution for several days. Paper Chromatography In order to test whether these fluorescences will be due to anthranilic acid or not, paper chromatographic test was performed. The results are shown in Fig. 1. It has been indicated that the fluorescence of the products in the medium containing peptone or the aromatic amine des- cribed above might be ascribable to their identity with anthranilic acid. On the 0 0 Anthranilic acid C o a b Fig. 1. Paper chromatography of culture extract of the bacteria incubated in the presence of inhibitory agent of pigmentation. a, Result with the same medium as No. 1 in Table 1 ; b, with the medium containing 0.05% acetanilide and the same components as in a. Solvent, n-butanol-acetic acid-water (4:1:2). (103) Mamoru KURACHI contrary, the fluorescence shown in the synthetic medium without peptone or inhibitory agents, could not be ascribed to anthranilic acid. It is worthy to note why anthranilic acid is detected in the peptone medium. This reason can not, of course, be attributed to its inhibitory action, but to its breaking down as will be illustrated later. It is an interesting fact that P-aminobenzoic acid which is to be an essential metabolite in the bacteria, competitively inhibits the metabolism of anthranilic acid. It has been believed by the author that the key to solve the mechanism of toxic action of this compound on the bacterial growth must be found in this point. Absorption Spectrum As a further study to ascertain the identity of the fluorescent product with anthranilic acid, a photometric test has been done. For the paper chromatography, the following solvent system was employed : n-butanol-acetic acid-water (4 : 1 : 2). Since the spot on the one-dimensional paper chromatogram using the above solvent system gave a mixed absorption spectrum of anthranilic acid and inhibitory agent, this eluate was again chromatographed with the solvent, benzene-alcohol (8:1). Fig. 2 represents the result in the case of acetanilide. As might have been expected, a characteristic absorption band was observed, suggesting that the accumulation product is identical with anthranilic acid itself. 1.4- 1.2 >, 1.0 - c 0.8 - 0 00.6 0.4 - 0.2 -°\e o/ o~ °\o\ o~ o\ 240 260 280 300 320 340 360 Wavelength (mp) Fig. 2. Absorption curve of a culture extract of the bacteria incubated in the presence of acetanilide. Medium was the same as in the case of a in Fig. 1. (e—•-1110) Anthranilic acid, (O—O—O) the culture extract. Absorbances were measured with neutral aqueous solution in each case. Formation of Anthranilic Acid by Catabolic Reaction As mentioned before, the formation of anthranilic acid in the culture medium prepared with peptone, has been assumed to originate from tryptophan which is to be a component of peptone. In order to verify this assumption the following experiment was carried out : 0.001M DL-tryptophan dissolved in O.O1M phosphate buffer, pH 7.6, was incubated at 3T for 12 hours, with resting bacterial cells suspended at a level of 0.02g wet cell per ml of the solution. (104) Studies on the Biosynthesis of Pyocyanine. (VIII) The paper chromatographic test has shown that tryptophan was converted to anthranilic acid by resting cells (Fig. 3). Indolca:;s'?"";a%` , Anthranilic( 0 0 fl f acid, PABA.j:W Tryptophan-.; Scrinc• a b c d e Tryptophan Indolc Anthranilic acid Fig. 3. Paper chromatograms of thereaction products from tryptophan, from indole and from anthranilic acid. a, Tryptophan ;

Load more